In the last decade, the properties of graphene materials have been intensively studied in the scientific community in order to develop their potential in various biomedical applications. Graphene is a single layer (in thickness) of carbon atoms arranged in hexagonal rings of an aromatic electron structure. Graphene oxide and reduced graphene oxide are the graphene derivatives characterized by the presence of oxygen functional groups on the graphene surface (more in graphene oxide (GO) than in reduced graphene oxide (RGO)) and, therefore, by the presence of defects in the graphene hexagonal rings as well. Such disorder in the graphene structure leads to many unique properties of GO and RGO.
Due to the presence of oxygen groups bonded to the surface, GO is an electrical insulator (ideal graphene is an excellent semiconductor). GO is highly hydrophilic and therefore has great wettability. The surface of both GO and RGO is susceptible to modifications with different molecules, including biological ones, which allows composites with strictly controlled and desired properties to be synthesized. GO and RGO, due to their large active surface areas, can serve as a platform for biological molecules (both organic and inorganic) to be safely introduced into the organism without the risk of uncontrolled and undesirable spreading into the surrounding tissues. This is a great advantage, for example, in the case of anticancer therapy, where cytotoxic compounds are introduced into the tumor [1
In many biomedical applications, it is crucial to use materials that are nontoxic to human cells, in terms of this requirement GO and slightly reduced GO seem to be suitable materials. The biocompatibility of these two graphene derivatives has been confirmed by many publications in the literature [6
]. This aspect of GO and RGO materials presented in this study was also previously investigated by us with human umbilical cord mesenchymal stem cells (hUC-MSCs) [7
]. The overriding conclusion from this study was that none of the tested materials (GO and RGO) were characterized with a negative impact in the cellular viability, proliferation, morphology, or gene expression. It was also found that the cytotoxicity depends on the size of the graphene flakes and the content of the oxygen functional groups: smaller flakes (about 0.2–2 μm of lateral size) and highly reduced graphene oxide (approximately 10% of remaining oxygen atoms) do have a toxic effect. Therefore, in this work we used RGO with a higher oxygen content and GO with larger flakes that were nontoxic to hUC-MSCs.
There are two main synthesis methods of graphene oxide: Hummers and Offeman [8
] and Marcano and Tour [9
]. The first one consisted of adding potassium manganate (KMnO4
) as an oxidant to the reaction mixture in the presence of concentrated sulfuric acid (VI) and NaNO3
salt. Marcano and Tour proposed an improved method of graphite oxidation synthesis involving the elimination of sodium nitrate (V) from the reaction mixture, increasing the amount of potassium manganate (VII) and carrying out the reaction in the presence of concentrated acids: H2
in a volume ratio of 9:1. As a result, GO with a higher content of oxygen functional groups can be obtained. On the basis of these methods, numerous modifications are made to the synthesis of graphene oxide: partial oxidation to prepare GO of C/O atomic ratio between 12 and 3 [10
], double oxidation leading to a high carboxyl groups concentration [11
]; the use of different concentration of nitric acid (from 50% to 98%) leading to GO structure of C/O atomic ratio between 6.5 and 2.8 [12
] and fast, 1-h oxidation [13
In this work, we used Ag, Ag2
O, Au, and TiO2
nanoparticles to create composites with GO and RGO. Silver nanoparticles (AgNPs) were chosen because of their antibacterial properties—AgNPs have been shown to be effective against gram-positive and gram-negative bacteria, as well as fungi and viruses. Currently, nanosilver is used in many fields of industry, most widely in the production of dressings for accelerated wound healing and medical implants, as well as in cosmetics, dentistry, water filtration, or textile production. The mechanism of its action is based on its ability to interact with the thiol groups of bacterial cell walls and to disrupt the cell membrane. This leads to the denaturation and inactivation of enzymatic proteins that are a part of the respiratory chain, the formation of reactive oxygen species (ROS) and, consequently, the appearance of oxidative stress of the cell [14
Gold nanoparticles (AuNPs) are widely used in such areas of medicine as diagnostics, cancer treatment and drug delivery systems. The ability of nanogold to enhance light scattering and absorption due to surface plasmon resonance is used in cancer diagnosis and therapy. Binding of nanogold to ligands allows the specific targeting of biomarkers for imaging and cancer detection. In addition, it can convert absorbed light into localized heat energy, which can be used in laser photothermal therapy. In addition, it has been shown that AuNPs can have a positive effect on the differentiation (osteogenesis) of hMSCs. The influence of AuNPs’ shape and size on the survival, proliferation, and expression of osteogenic gene markers has been demonstrated [15
Nano-titanium oxide, in addition to its antibacterial properties which have been used in decontamination preparations, is widely used in tissue engineering as part of biocomposites and surface coatings. The topography of titanium’s surface plays a very important role in biomedical applications. As the rough and porous morphology of Ti imitates native bone architecture, it enhances osteoblasts adhesion, maturation and bone formation. The distribution of charge and the surface chemistry of titanium materials are also important and can be one of the key factors inducing stem cells differentiation to osteoblasts. Much work has focused on the use of nano-TiO2
as a photosensitizer in the treatment of cancer due to its high photocatalytic activity, low toxicity, and high photostability [18
Literature reports indicate the possibility of using nano-Ag2
O in the treatment of venous ulceration. Silver oxide used as an ingredient in dressing ointment resulted in improved microcirculation and wound healing [20
]. Considering the possible anti-inflammatory properties and accelerated wound healing, we also decided to prepare a nano-Ag2
O composite with flake graphene as a potential material in tissue engineering.
The other approaches related to the fabrication of the antibacterial composites based on the graphene oxide contained the addition of graphitic carbon nitride (GO/g-C3
], zinc oxide (ZnO) [22
] and Ag/Cu bimetallic nanoparticles (NPs) [23
]. Other kinds of GO composites are those with the anti-inflammatory properties. They were formed using Fe2
], aerogel (GA)-supported metal-organic framework (MOF) particles [25
] and polyoxotungstate [26
]. There is also another great research area that uses graphene composites: regenerative tissue engineering. The examples can be calcium silicate—graphene composites [27
] and graphene oxide-calcium phosphate nanocomposites [28
] for osteogenic and angiogenic differentiation of human mesenchymal stem cells. For neural tissue engineering, RGO is commonly used due to its electrical properties and one of the described materials is RGO/TiO2
for photo stimulation of neural stem cells [29
Knowing that both morphology and chemical composition of composites for potential biomedical applications may result in different properties of the obtained materials, we decided to perform a detailed and systematic study addressing this issue. For this purpose, this manuscript is focused exclusively on the material aspect: the synthesis and characterization of graphene-nanoparticles composites. This work, for the first time, provides a comprehensive study concerning both GO and RGO composites with various types of inorganic nanoparticles of a specified biological activity. We plan to perform and discuss the biological assessment of the aforementioned composite materials in the future.
2. Materials and Methods
For the experiments, the following chemical compounds were used: graphite (Asbury Carbons, Asbury, NJ, U.S.; with a particle diameter of 300–425 µm), sulfuric acid (Poch S.A., Gliwice, Poland, 96–98% pure p. a.), orthophosphoric acid (Chempur, Piekary Śląskie, Poland, pure p. a.), potassium permanganate (Chempur, Poland, pure p. a.), L(+)-ascorbic acid (Poch S.A, pure p. a.), sodium hypophosphite monohydrate (Chempur, pure p. a.), hydrochloric acid (Chempur, pure p. a.), perhydrol (Chempur, pure p. a.), and ethanol (Poch S.A., 96%, pure p. a.). The compounds used for nanoparticle synthesis included NaOH (Chempur, pure p. a.), NaBH4 (Sigma Aldrich, Schnelldorf, Germany, 99%), Polyphenon PP60 (Sigma Aldrich), AgNO3 (pure p. a.), HAuCl4·3H2O (Roth, ≥ 99.5%), titanium isopropoxide (Sigma Aldrich, 97%), sodium citrate (Poch S.A., pure), and absolute ethanol (Merck Millipore, Darmstadt, Germany).
The morphology of the materials was examined by scanning electron microscopy SEM (Auriga CrossBeam Workstation, Carl Zeiss) and atomic force microscopy AFM (Dimension Icon, Bruker; with tapping mode and OTESPA R3 scanning probe, Bruker). The chemical structure was studied with Raman Spectroscopy (Renishaw Invia, excitation laser source: 532 nm; laser power: < 1 mW), XPS Spectroscopy (UHV Multichamber XPS System, Prevac; with Al Kα X-ray source (1486.6 eV)) and X-ray diffraction XRD (Rigaku Diffractometer, Japan; with Cu Kα anode of 8.038 keV, UC=C40 kV, IC=C30 mA, scanning speed: 2 deg/min, sampling density: 0.02 deg). For XRD, Raman and XPS measurements, the samples were prepared in the form of powders. AFM, SEM and EDS measurements were conducted on layers of the samples placed on a silicon substrate, without sputtering.
2.1. GO Preparation
The GO was prepared by the modified Marcano method [9
]. In brief: 3 g of graphite flakes (with the average size of 300–425 µm) were added gradually to a reactor containing 360 mL of concentrated sulphuric acid and 40 mL of orthophosphoric acid. After that, 18 g of potassium permanganate were slowly added to the graphite. The oxidation process was conducted for a few hours and it was stopped by the addition of deionized water and finally—3 mL of perhydrol (30% H2
; Chempur, pure p.a.). The water suspension of such obtained graphite oxide was left to sediment. The purifying process was carried out with a custom made microfiltration system. Due to specific shearing forces acting on the GO flakes during the purification process, the flakes were exfoliated at the same time.
The chemical formula of GO was assumed as “C2
O”, where there is one oxygen atom per two carbon atoms, which gives a molar mass of 40 g/mol. Such structure is in agreement with the information obtained from the XPS measurement and with the literature as well [30
2.2. RGO Preparation
The RGO was prepared via a “green” reduction process by using L(+)-ascorbic acid (C6
) as a reducing agent. An L-ascorbic acid solution was added to the previously prepared GO water suspension with GO: L-ascorbic acid molar ratio of 1:4. The mixture was reduced for 3.5 h at a temperature of 95 °C with constant stirring. The GO molar mass was assumed to be 40 g/mol with respect to the C2
O chemical formula. The prepared RGO was then filtered with ultrapure water to remove the remaining ions. Gao et al. (2010) postulated a reduction mechanism proceeded via a two-step SN
2 nucleophilic reaction followed by another one-step thermal elimination [31
2.3. Preparation of Graphene Composites with Ag, Au, Ag2O, and TiO2 Nanoparticles
2.3.1. Composites with Nano-Ag
Three different reducing agents were used to reduce Ag+ ions to Ag nanoparticles: L(+) ascorbic acid (Ag[KA]), sodium borohydride (Ag[BS]) and polyphenols from green tea (Ag[PP60]).
Composites with Nano-Ag[KA]
To prepare the composite of GO with nano-Ag obtained with L-ascorbic acid (GO Ag[KA]), a silver nitrate (V) solution was added to the aqueous suspension of GO while stirring and after a few minutes an aqueous solution of L-ascorbic acid was added dropwise. The reduction mechanism is shown in Figure 1
. The molar ratio of GO to silver nitrate (V) was 1:0.08, while the molar ratio of silver nitrate (V) to L-ascorbic acid was equal to 1:2. Adding the compounds in this order resulted in a homogeneous coating of graphene oxide with silver particles. The reaction was carried out at room temperature to ensure that the reduction would involve silver ions only, without disturbing the graphene oxide structure. The mixture was left for 24 h with vigorous stirring and was then dialyzed for 72 h to remove the residual ions.
The RGO composite with Ag[KA] (RGO-Ag[KA]) was prepared using the above recipe for GO-Ag[KA], followed by the addition of L-ascorbic acid to finally reduce the GO. The reaction was performed for 3.5 h at 95 °C. The GO: L-ascorbic acid molar ratio was 1:4. After the reaction was completed, the material was purified by dialysis. The two-step process was carried out to obtain a good distribution of silver nanoparticles on RGO flakes. Because GO flakes are well dispersed, firstly, nano-Ag was precipitated on these non-agglomerated flakes. After that, the GO reduction causing also partial agglomeration of the flakes was conducted (with the use of ascorbic acid and the higher temperature) without negative influence on the distribution of the nanoparticles on RGO flakes.
Composites with Nano-Ag[BS]
A reaction between silver nitrate and sodium borohydride in a water solution was described by Sobczak-Kupiec et al. (2011) [32
]. In brief, the reaction can be written as follows:
AgNO3 + NaBH4 + 3H2O → Ag + NaNO3 + H3BO3 + 3.5H2
To obtain the GO-Ag [BS] composite, silver nitrate (V) was added successively to the aqueous GO suspension, followed by sodium borohydride. The molar ratio of GO (MC=C40 g/mol) to silver was 1:0.08 and the molar ratio of silver nitrate (V) to sodium borohydride was 1:2. NaBH4
creates a reductive and alkaline environment, causing deprotonation of carboxylic groups, which renders more negative zeta potential and therefore improves the GO flakes stability [33
]. However, salt type and ionic strength have a significant effect on GO stability. Here, the presence of Na+
ions could compensate for the effect of the decrease in zeta potential, finally leading to the flakes agglomeration. The hydrodynamic dimension depends on the type of ions present in the GO sample and is bigger for multivalent ions than monovalent ones; it also depends on the concentration of these ions [34
The reaction was continued for 24 h at a room temperature with continuous stirring. The material was then dialyzed for 72 h to remove the remaining ions.
The RGO-Ag[BS] composite was obtained through the preparation of the GO-Ag[BS] composite followed by the addition of L-ascorbic acid to reduce the GO. The reaction was carried out for 3.5 h at 95 °C. The GO: L-ascorbic acid molar ratio was 1:4. After the reaction was completed, the material was dialyzed for 72 h.
Composites with Nano-Ag[PP60]
To obtain the GO-Ag[PP60] composite, an aqueous AgNO3 solution was added to the aqueous suspension of GO at a molar ratio of 1:0.08. Then, the polyphenol PP60 solution was added dropwise in a 1:1 molar ratio with AgNO3. The reagents were vigorously stirred on a magnetic stirrer for 24 h at room temperature. The material was purified by dialysis.
The procedure for obtaining the RGO composite with Ag[PP60] nanoparticles consisted of producing the GO-Ag[PP60] composite (as described above) and adding L-ascorbic acid to reduce the GO. The reaction was carried out for 3.5 h at 95 °C. The GO:ascorbic acid molar ratio was 1:4. After the reaction was completed, the material was dialyzed.
2.3.2. Composites with Nano-Au
To prepare the GO-Au composite, an aqueous solution of HAuCl4·3H2O was added dropwise to the GO water suspension at a GO:HAuCl4 molar ratio of 1:0.08. The mixture was stirred on a magnetic stirrer for 30 min at room temperature (to obtain a homogeneous mixture of GO flakes and Au3+ ions), after which the temperature was increased to 80 °C to provide the conditions needed for Au3+ reduction. Then, an aqueous solution of sodium citrate (3-hydrated) was added dropwise at a molar ratio with HAuCl4 of 1:0.17. The reaction continued for 1 h at 80 °C while being stirred. A composite with a purple glow was formed, proving the formation of gold nanoparticles. The material was dialyzed to remove the remaining ions. The mild reaction conditions did not reduce the GO, only Au3+ to Au0.
To prepare the RGO-Au composite, L-ascorbic acid was added to the previously prepared GO-Au composite. The reduction was carried out on a magnetic stirrer for 3.5 h at 95 °C and purified by dialysis.
2.3.3. Composites with Nano-Ag2O
The GO-Ag2O composite was obtained by adding silver nitrate (V) solution followed by sodium hydroxide solution to the aqueous GO suspension. This order of adding compounds was used because by adding NaOH first would cause GO agglomerates to form (in the highly alkaline environment) and the Ag nanoparticles to be poorly distributed on the flakes. The addition of AgNO3 to GO before NaOH provides less alkaline conditions due to the presence of strong acid salt. The molar ratio of GO to silver nitrate (V) was 1:0.08 and the molar ratio of silver nitrate (V) to sodium hydroxide was 1:1. The reaction was carried out at room temperature for 24 h with continuous stirring. The resulting material formed a stable aqueous suspension that was dialyzed to remove residual ions.
O composite was produced by reducing GO with L-ascorbic acid as described in Section 2.2
. An aqueous solution of AgNO3
and NaOH was then added to RGO to precipitate Ag2
O nanoparticles. The reactions were carried out at room temperature for 24 h followed by purification with dialysis.
2.3.4. Composites with Nano-TiO2
To obtain the GO-TiO2 composite, titanium isopropoxide was added dropwise to a small amount of absolute ethanol and then added slowly to the aqueous GO suspension under vigorous stirring. During the addition of GO, a white TiO2 precipitate formed. Proper amounts of the compounds were used to obtain a molar GO:TiO2 ratio of 1:0.08.
The RGO-TiO2 composite was prepared by reducing the GO with L-ascorbic acid and purifying it by dialysis. The appropriate amount of titanium isopropoxide (standard 1:0.08 molar ratio) was then added dropwise to a small amount of absolute ethanol, after which it was added slowly to the RGO suspension under vigorous stirring. The sample was stirred for 24 h in order to precipitate out TiO2 particles.